Novel gene encoding formate dehydrogenases d &amp; e and method for preparing succinic acid using the same

ABSTRACT

Nucleotide sequences encoding formate dehydrogenases D &amp; E and a method for preparing succinic acid using the same, more particularly, formate dehydrogenases D &amp; E converting formate to carbon dioxide and hydrogen, fdhD and fdhE nucleotide sequences encoding the formate dehydrogenases D &amp; E, recombinant vectors containing the nucleotide sequences, microorganisms transformed with the recombinant vectors, and a method for preparing succinic acid using the transformed microorganism.

CROSS-REFERENCE TO RELATED APPLICATION

This is a continuation of U.S. patent application Ser. No. 11/228,945filed Sep. 16, 2005 and claims the priority of such earlier applicationunder 35 USC 120. U.S. patent application Ser. No. 11/228,945 in turnclaims the priority under 35 USC 119 of Korean Patent Application No.10-2005-0076348 filed on Aug. 19, 2005 in the Korean IntellectualProperty Office. The disclosure of said U.S. patent application Ser. No.11/228,945 and said Korean Patent Application are hereby incorporatedherein by reference in their respective entireties.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to novel genes encoding formatedehydrogenases D & E and to a method for preparing succinic acid usingthe same, more particularly, to formate dehydrogenases D & E convertingformate to carbon dioxide and hydrogen, novel fdhD and fdhE genesencoding the formate dehydrogenases D & E, a recombinant vectorcontaining the genes, a microorganism transformed with the recombinantvector, and a method for preparing succinic acid using the transformedmicroorganism.

2. Background of the Related Art

Succinic acid, which is a dicarboxylic acid (HOOCCH₂CH₂COOH) with fourcarbon atoms initially purified from amber resin, is used in a very widerange of industrial applications (Zeikus et al., Appl. Microbiol.Biotechnol., 51:545, 1999). Particularly, as the utility of succinicacid as a main raw material of biodegradable polymers was recentlyproven, a rapid increase in the demand of succinic acid is expected(Willke et al., Appl. Microbiol. Biotechnol., 66:131, 2004).

Succinic acid can be produced by chemical synthesis and fermentation.Most commercially available succinic acid recently has been producedfrom n-butane as a starting material derived from LNG or crudepetroleum, by chemical manufacturers, such as BASF, DuPont and BPChemicals. Chemical processes for the synthesis of succinic acid havethe problem that they cause the discharge of large amounts of harmfulsolid wastes, waste solutions and waste gases (including carbonmonoxide) during the preparation of succinic acid, and particularly,have the limitation that they use fossil raw material as a basicmaterial. Only a small amount of succinic acid, which is used in specialapplications, such as medical drugs, is currently produced bytraditional microbial processes.

In an attempt to solve the described problems occurring in the chemicalprocesses for the synthesis of succinic acid, studies on the productionof succinic acid by fermentation processes have been conducted by manyresearchers. The method for the production of succinic acid byfermentation is a method of producing succinic acid from renewable rawmaterials using microorganisms. Bacterial strains that are used in theproduction of succinic acid can be broadly divided into recombinant E.coli and ruminal bacteria, such as Actinobacillus, Anaerobiospirillum,Bacteroides, Mannheimia, Succinimonas, Succinivibrio, etc.

A research team of the University of Chicago has attempted to increasethe production of succinic acid by preparing a mutant strain AFP111(ATCC No. 202021) in which E. coli ldh and pfl genes involved in theproduction of lactic acid and formic acid have been removed and a ptsGgene of the glucose transfer system has been manipulated (U.S. Pat. No.5,770,435).

Among ruminal bacteria, Actinobacillus, Anaerobiospirillum andMannheimia strains have been relatively much-studied. MichiganBiotechnology Institute (MBI) has developed an Actinobacillussuccinogenes 130Z strain (ATCC No. 55618) and a process for producing ahigh concentration of succinic acid using the same (U.S. Pat. No.5,504,004). Also, such institute has developed Anaerobiospirillumsucciniciproducens and its mutant strains, and a process for theproduction and purification of succinic acid (U.S. Pat. Nos. 5,521,075;5,168,055; and 5,143,834).

However, the processes for preparing succinic acid using the describedstrains have low productivity and result in the production of largeamounts of byproducts in addition to succinic acid, thus requiring highcosts for the separation and purification of succinic acid. Accordingly,there has been an urgent need for the development of a bacterial systemthat has high productivity and at the same time, can inhibit theproduction of byproducts (Hong et al., Biotechnol. Lett., 22:871, 2000).

For this purpose, the isolation of an excellent succinic acid-producingbacterial strain, the establishment of genome sequences and theunderstanding of metabolic characteristics of bacterial strains based onthem are first required. With such basis, it then is necessary to securegene manipulation technologies required for the construction of a novelgene recombinant bacterial strain. Although there has been a priorattempt to increase the production of succinic acid using thephosphoenolpyruvate carboxykinase (pckA) gene of Anaerobiospirillumsucciniciproducens (Laivenieks et al., Appl. Environ. Microbiol.,63:2273, 1997), the art has failed to develop a gene recombinant strainbased on the full genome sequence of ruminal bacteria.

Meanwhile, the present inventors previously isolated a Mannheimiasucciniciproducens MBEL55E strain from the rumen of a Korea cow thatproduces succinic acid in high efficiency using various substrates, andreported the full genome sequence of the strain (Hong et al., NatureBiotechnol., 22:1275, 2004). Particularly, the above strain ischaracterized by immobilizing carbon dioxide, known as a greenhouse gas,in the synthesis of succinic acid. Also, this applicant previouslyprepared succinic acid with high yield by deleting a lactic aciddehydrogenase gene (ldhA) and a pyruvate formate-lyase (pfl) fromMannheimia succiniciproducens MBEL55E, so as to construct mutant strainMannheimia sp. LPK (KCTC 10558BP), and deleting a phosphotransacetylasegene (pta), and an acetate kinase gene (ackA) from the LPK strain toconstruct mutant strains Mannheimia sp. LPK7, and then culturing theresulting mutant strain in an anaerobic condition (WO 05/052135 A1).However, the mutant strain has a problem that it results in theaccumulation of formate to a certain degree as a byproduct during theculture thereof.

Accordingly, there continues to be an urgent need in the art for thedevelopment of a bacterial system for high productivity, low byproductsuccinic acid production that overcomes the deficiencies of the priorart.

SUMMARY OF THE INVENTION

The present invention relates to novel genes encoding formatedehydrogenases D & E derived from Mannheimia succiniciproducens MBEL55Ethat is usefully employed in the production of succinic acid.

The present invention relates on one aspect to a recombinant vectorcontaining said gene, and a recombinant microorganism transformed withsaid recombinant vector.

Still another aspect of the present invention relates to a method forpreparing succinic acid using said recombinant microorganism.

In one aspect, the present invention relates to formate dehydrogenases D& E having amino acid sequences of SEQ ID NOs: 7 and 8, respectivelywhich have the activities of converting formate to carbon dioxide andhydrogen, as well as genes (fdhD and fdhE) encoding the formatedehydrogenases D & E. In one preferred aspect of the present invention,said genes preferably have DNA sequences of SEQ ID NOs: 5 and 6.

In another aspect, the present invention relates to a recombinant vectorcontaining the fdhD and/or fdhE gene. In still another aspect, thepresent invention relates to a recombinant microorganism obtained byintroducing the fdhD and/or fdhE gene or the recombinant vector into ahost cell selected from the group consisting of bacteria, yeast andmold.

In a still further aspect of the present invention, the recombinantvector is preferably pMVDfdhDE, pMV19fdhDE, or pMExfdhDE, but is notlimited thereto. Additionally, the host cell is a succinicacid-producing microorganism, a lactic acid-producing microorganism, oran ethanol-producing microorganism. The succinic acid-producingmicroorganism is the genus Mannheimia microorganism, and preferably, aMannheimia succiniciproducens MBEL55E.

As shown in a succinate synthesis pathway described more fullyhereinafter with reference to FIG. 1, the fdhD and fdhE genes canconvert formate to CO₂ and H₂. Thus, it is possible to minimize formatewhich is produced as a byproduct in the production of succinic acid.Also, the reducing power (NADH) required for the synthesis of asuccinate is conferred, as well as, produced CO₂ increases theproduction of succinic acid by promoting the conversion of pyruvate tomalate and conversion of phosphoenolpyruvate to oxaloacetate, which isimportant in a succinate synthesis pathway.

Accordingly, the present invention relates in another aspect to a methodfor preparing succinic acid, the method including the steps of:culturing the recombinant microorganism; and recovering succinic acidfrom the culture broth of the recombinant microorganism. The steps ofculturing the recombinant microorganism and recovering the succinic acidcan be carried out by the culture method and the isolation andpurification method of succinic acid, which are generally known in theprior fermentation industry.

The inventive fdhD and fdhE genes can decrease the accumulation offormate in the production of acetic acid, lactic acid or ethanol. Thus,when acetic acid-, lactic acid- or ethanol-producing microorganism istransformed with the genes according to the present invention, theproduction of formate as a byproduct can be reduced remarkably.Accordingly, the present invention provides a method for preparingacetic acid, lactic acid or ethanol, the method comprising the steps of:culturing the lactic acid- or ethanol-producing microorganismtransformed with the fdhD and/or fdhE gene; and recovering acetic acid,lactic acid or ethanol from the microbial culture broth.

The above and other features and embodiments of the present inventionwill be more fully apparent from the following detailed description andappended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram showing a pathway for the synthesis ofsuccinic acid from Mannheimia strain.

FIG. 2 is a gene map of recombinant plasmid pMExfdhDE.

FIG. 3 is an SDS-PAGE showing the protein expression of recombinantMannheimia MBEL55EpMExfdhDE containing recombinant plasmid pMExfdhDE.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

The present invention is based on the discovery of a bacterial systemfor high productivity, low byproduct succinic acid production thatovercomes the deficiencies of the prior art.

The present inventors have made extensive efforts to find the core geneinvolved in succinic acid metabolism in order to develop a microbialstrain capable of minimizing the production of formate and of producingsuccinic acid with higher yield, on the basis of a succinic acidsynthetic pathway shown in FIG. 1, and as a result, they have clonedformate dehydrogenase D & E-encoding genes (fdhD and fdhE) derived fromMannheimia succiniciproducens MBEL55E and determined the functionthereof, thereby completing the present invention.

The present invention is more fully described hereinafter and withreference to illustrative examples. It is to be understood, however,that these examples are presented in order to more fully describe thepresent invention, and are correspondingly not intended to be construedto limit the present invention.

Although only the use of the specified expression vector and the genusMannheimia microorganism, as a host cell, to express the inventive gene,is illustrated in the following examples, the use of other kinds ofexpression vectors and succinic acid-producing microorganisms will bereadily apparent to those skilled in the art. Also, it will be readilyapparent to a person skilled in the art that the known aceticacid-producing microorganism, lactic acid-producing microorganism andethanol-producing microorganism in place of the succinic acid-producingmicroorganism can be used as a host cell.

Example 1 Preparation of Mannheimia/E. Coli Shuttle and ExpressionVector pMEx

Mannheimia/E. coli shuttle vector pMEx was prepared from pMVSCS1reported to be isolated from Mannheimia (Kehrenberg et al., J.Antimicrob. Chemother., 49:383, 2002) and E. coli expression vectorpKK223-3 (Amersham Pharmacia Biotech). For this purpose, pKK223-3 waspartially digested with BamHI and AccI to collect a 2.7 kb fragmentcontaining pBR322 ori and an ampicillin-resistant gene, and the singlestrand portions are filled with T4 DNA polymerase to make blunt ends.The blunt ends are ligated to prepare pKKD (2.7 kb). pMVSCS1 (5.6 kb)was digested with XhoII, and ligated with pKKD digested with restrictionenzyme BamHI to prepare fusion vector pMVD (8.3 kb). The pMVD wasdigested with NcoI, and a 5.9 kb fragment was religated to constructMannheimia/E. coli shuttle vector pME.

The pME was digested with BamHl and Clal, and a promoter and atranscription termination sequence of Mannheimia pckA gene (Hong et al.,Nature Biotechnol., 22:1275, 2004) were amplified to clone on the samerestriction enzyme site whereby pMEx was constructed.

Example 2 Identification of Novel Genes (fdhD and fdhE) Derived fromMannheimia succiniciproducens MBEL55E and Preparation of a RecombinantPlasmid Introduced with fdhDE Genes

The fahD and fahE genes having SEQ ID NOs: 5 and 6, which encodesformate dehydrogenase D & E derived from Mannheimia succiniciproducensMBEL55E (KCTC 0769BP) were cloned, including a promoter and atranscription termination sequence, respectively.

For this purpose, the chromosome of Mannheimia succiniciproducensMBEL55E as a template was subjected to PCR with primers of SEQ ID NOs: 1and 2 for fdhD and primers of SEQ ID NOs: 3 and 4 for fdhE underconditions shown in Table 1 below. The resulting fdhD and fdhE geneswere amplified. fdhD was digested with restriction enzyme BamHI andligated to Mannheimia/E. coli shuttle vector pMEx digested with the samerestriction enzyme to construct plasmid pMExfdhD. The pMExfdhD wasdigested with EcoRl and ligated to fdhE digested with the samerestriction enzyme to construct plasmid pMExfdhDE (FIG. 2). In this way,a formate dehydrogenase D & E-encoding genes (fdhD and fdhE) derivedfrom Mannheimia succiniciproducens MBEL55E were cloned. TABLE 1Conditions for amplification of fdhD and fdhE genes. Restriction enzymesite contained in Gene Primer the primer Reaction condition fdhDfdhD-F(SEQ ID NO: 1) BamHI Cycle I: 94° C., 5 min fdhD-R(SEQ ID NO: 2)Cycle II: (30 cycles) 94° C., 40 sec 56° C., 30 sec 72° C., 3 min CycleIII: 72° C., 5 min Cycle IV: 4° C., store fdhE fdhE-F(SEQ ID NO: 3),EcoRII Cycle I: 94° C., 5 min fdhE-R(SEQ ID NO: 4) Cycle II: (30 cycles)94° C., 40 sec 56° C., 30 sec 72° C., 3 min Cycle III: 72° C., 5 minCycle IV: 4° C., store

The DNA sequences of the cloned fdhD and fdhE of Mannheimiasucciniciproducens MBEL55E were analyzed and the amino acid sequences offormate dehydrogenases D & E were inferred. As a result, the fdhD andfdhE genes of Mannheimia succiniciproducens MBEL55E had DNA sequences of846 bp (SEQ ID NO: 5) and 984 bp (SEQ ID NO: 6), and the formatedehydrogenases D & E consisted of 281 amino acid residues (SEQ ID NO: 7)and 328 amino acid residues (SEQ ID NO: 8), respectively.

The conserved domains of the fdhD and fdhE amino acid sequences derivedfrom Mannheimia succiniciproducens MBEL55E were analyzed, and as aresult, these genes showed high homology (fdhD score: 280, fdhE score:376) with fdhD/NarQ family (gnl|CDD|3141, pfam02634) and fdhE(gnl|CDD|9775, pfam04216). The G+C amount of the Mannheimiasucciniciproducens MBEL55E fdhD and fdhE were 42.0% and 40.8%,respectively.

Meanwhile, the frequency of using amino acid codons in the Mannheimiasucciniciproducens MBEL55E fdhD and fdhE genes were examined and theresults are shown in Table 2 below. As shown in Table 2 below, thefrequency of using amino acid codons in the fdhD and fdhE genes showeddifferent result from that in generally known E. coli. For example, forthe frequency of using lysine codons, AAAs were used at frequencies of89% and 75% in the Mannheimia succiniciproducens MBEL55E fdhD and fdhEgenes, respectively, but AAA was used at a frequency of 76% in generallyknown E. coli. For the frequency of using glutamate codons, GAAs wereused at high frequencies of 82% and 88% in the Mannheimiasucciniciproducens MBEL55E fdhD and fdhE genes, respectively, but GAA inE. coli was used at a frequency of 70%. Also, for the frequency of usingglutamine codons, CAAs were used at high frequencies of 69% and 87% inthe Mannheimia succiniciproducens MBEL55E fdhD and fdhE genes,respectively, but at a frequency of 31% in E. coli. TABLE 2 Frequency ofusing amino acid codons Frequency Frequency Average Frequency FrequencyAverage of use in of use in frequency of use in of use in frequencyAmino MBEL55E MBEL55E of use in Amino MBEL55E MBEL55E of use in acidCodon fdhD fdhE E. coli acid Codon fdhD fdhE E. coli Ala GCA 0.27 0.270.22 Leu CTA 0.06 0.06 0.03 GCC 0.14 0.17 0.25 CTC 0.03 0.06 0.10 GCG0.50 0.33 0.34 CTG 0.06 0.11 0.55 GCT 0.09 0.23 0.19 CTT 0.10 0.33 0.10Arg AGA 0.31 0.00 0.04 TTA 0.48 0.31 0.11 AGG 0.00 0.00 0.03 TTG 0.260.14 0.11 CGA 0.13 0.38 0.05 Lys AAA 0.89 0.75 0.76 CGC 0.25 0.31 0.37AAG 0.11 0.25 0.24 CGG 0.13 0.08 0.08 Met ATG 1.00 1.00 1.00 CGT 0.190.23 0.42 Phe TTC 0.21 0.46 0.49 Asn AAC 0.43 0.14 0.61 TTT 0.79 0.540.51 AAT 0.57 0.86 0.39 Pro CCA 0.00 0.29 0.20 Asp GAC 0.23 0.14 0.41CCC 0.25 0.14 0.10 GAT 0.77 0.86 0.59 CCG 0.25 0.21 0.55 Cys TGC 0.380.44 0.57 CCT 0.50 0.36 0.16 TGT 0.63 0.56 0.43 Ser AGC 0.13 0.26 0.27STOP TAA — — 0.62 AGT 0.27 0.26 0.13 TAG — — 0.09 TCA 0.33 0.04 0.12 TGA— — 0.30 TCC 0.00 0.11 0.17 Gln CAA 0.69 0.87 0.31 TCG 0.13 0.19 0.13CAG 0.31 0.13 0.69 TCT 0.13 0.15 0.19 Glu GAA 0.82 0.88 0.70 Thr ACA0.25 0.50 0.12 GAG 0.18 0.13 0.30 ACC 0.21 0.14 0.43 Gly GGA 0.14 0.250.09 ACG 0.33 0.07 0.23 GGC 0.29 0.25 0.40 ACT 0.21 0.29 0.21 GGG 0.190.00 0.13 Trp TGG 1.00 1.00 1.00 GGT 0.38 0.50 0.38 Tyr TAC 0.50 0.330.47 His CAC 0.00 0.18 0.48 TAT 0.50 0.67 0.53 CAT 1.00 0.82 0.52 ValGTA 0.24 0.36 0.17 Ile ATA 0.29 0.20 0.07 GTC 0.10 0.07 0.20 ATC 0.180.20 0.46 GTG 0.38 0.14 0.34 ATT 0.53 0.60 0.47 GTT 0.29 0.43 0.29

Example 3 Production of Succinic Acid by Use of Transformed Mannheimia

The recombinant plasmid pMExfdhDE constructed in Example 2 wastransformed into Mannheimia succiniciproducens MBEL55E byelectroporation to prepare MBEL55EpMExfdhDE. Also, pMEx was introducedinto Mannheimia succiniciproducens MBEL55E, to prepare MBEL55EpMEx.

Each of the prepared recombinant strains was inoculated in 10 ml of acomplex medium containing 9 g/l of glucose and cultured in an anaerobiccondition at 39° C. for 16 hours. Each of the cultured strains wastransferred in 250 ml of a complex medium containing 9 g/l of glucoseand further cultured in the medium at 39° C. At this time, 100 μg/l ofampicillin as an antibiotic was added. The fermentation of each of thestrains was performed by inoculating 250 ml of the Mannheimia culturebroth in 2.5 L of a complex medium, and the fermentation conditions wereas follows: initial glucose concentration: 20 g/l, pH: 6.8, and culturetemperature: 39° C. For the adjustment of pH during the fermentation,ammonia solution (28%, v/v) was used, and the concentration ofantibiotic ampicillin was the same as described above. A sample fromeach of the recombinant Mannheimia strains was collected during thefermentation, and the collected sample was centrifuged at 13,000 rpm and4° C. for 10 minutes, and the concentrations of metabolites and succinicacid in the supernatant were analyzed by high-performance liquidchromatography (HPLC). The results are shown in Table 3 below.

As shown in Table 3, in the case where the recombinant plasmid pMExfdhDEcontaining the fdhD and fdhE genes was introduced into the Mannheimiasucciniciproducens MBEL55E, the concentration of formate was reduced.These results suggest that the fdhD and fdhE genes encode an enzymeconferring an important reduction power on the step of producingsuccinic acid from fumarate in the succinic acid-producing pathway bysynthesis of NADH. TABLE 3 Productions of succinic acid usingtransformed Mannheimia Cell Formate Formate Succinic acid Fermentationconcentration concentration reduction concentration Strain Plasmid time(hrs.) (OD₆₀₀) (g/l) rate (%) (g/l) MBEL55E pMEx 8 7.2 4.66 100 10.4MBEL55E pMExfdhDE 10 6.5 2.03 156 9.4

Meanwhile, each of the strains was analyzed by SDS-PAGE, and the resultsare shown in FIG. 3. As can be seen in FIG. 3, the recombinantMannheimia succiniciproducens MBEL55EpMExfdhDE transformed with therecombinant plasmid pMExfdhDE showed a remarkable increase in theexpression of formate dehydrogenases D & E as compared to therecombinant Mannheimia succiniciproducens MBEL55EpMEx (control group)transformed with pMEx.

Example 4 Measurement of Formate Dehydrogenase D & E Activities

The culture broth of the recombinant Mannheimia succiniciproducensMBEL55EpMExfdhDE prepared in Example 3 was centrifuged at 13,000 rpm and4° C. for 5 minutes. The precipitated cells were washed 2 times with aniced buffer solution (100 mM Tris-HCl (pH 7.0), 20 mM KCl, 5 mM MnSO₄, 2mM DTT, 0.1 mM EDTA), and the washed cells were suspended in the samebuffer and the cell membranes were disrupted by sonication. The celldebris was removed by a centrifugation, and the cell extract supernatantwas used for the measurement of enzyme activities.

The enzyme activity of the cell extracts was measured with aspectrophotometer, in which the cell extract was allowed to react byadding a reaction buffer (200 mM sodium formate, 2 mM NAD+ and 100 mMpotassium phosphate buffer, pH 6.5) to a 1 cm-width cuvette and addingthe cell extract to the reaction buffer to a final volume of 1 ml, andthe NADH at 340 nm was measured. The results are shown in Table 4.

As shown in Table 4, the MBEL55EpMExfdhDE cell extract showed 305%increase in enzyme activity compared to the MBEL55EpMEx cell extract.This result confirms that the fdhD and fdhE genes according to thepresent invention are genes encoding formate dehydrogenases D & E havingthe activity of converting formate to carbon dioxide and hydrogen. TABLE4 Enzyme activities of transformed Mannheimia strains Enzyme activityStrain Plasmid *Enzyme activity (U) increase (%) MBEL55E pMEx 3.6 100MBEL55E pMExfdhDE 11.0 305*Enzyme activity shows the titer of formate dehydrogenase contained in 1mg of total protein. An enzyme activity of 1.0 U is defined as theamount of enzyme required for converting 1 nmole of a substrate to acertain product at 37° C. for 1 minute.

In the measurement of enzyme activity according to the presentinvention, it was identified that the reduction of NAD+produced NADH andcarbon dioxide. Thus, the formate dehydrogenases D & E confer toreducing power (NADH) required for the synthesis of a succinate,moreover CO₂ produced from the formate dehydrogenases D & E is useful inthe conversion of pyruvate to malate and conversion ofphosphoenolpyruvate to oxaloacetate, which is important in a succinatesynthesis pathway.

The activity of the formate dehydrogenases according to the presentinvention was compared to the known enzyme, and the result is shown inTable 5 below. As shown in Table 5, the formate dehydrogenases of theMannheimia strain transformed with the inventive fdhD and fdhE genesshowed much higher activity than the formate dehydrogenase of E. coliK12 (Gray et al., Biochim. Biophys. Acta, 117:33, 1966). TABLE 5Comparison of formate dehydrogenase activity between transformedMannheimia and E. coli Strain Enzyme activity (U) Gene homology (%)MBEL55EpMExfdhDE 11 60.1 (fdhD) 57.0 (fdhE) E. coli K12 5

As described and proven in detail above, the present invention providesthe novel genes (fdhD and fdhE) encoding the formate dehydrogenases D &E. The nucleotide sequences of the fdhD and fdhE genes may be DNAsequences of SEQ ID NO: 5 and SEQ ID NO: 6, respectively, or sequenceshaving appropriate homology thereto (e.g., that is at least 85%, andmore preferably is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%homologous to the nucleotide sequences of SEQ ID NO: 5 and SEQ ID NO:6). The novel genes according to the present invention are useful toprepare a recombinant microorganism capable of effectively reducingformate which is produced as a byproduct in the production of succinicacid, as well as conferring reducing power (NADH) required for thesynthesis of succinic acid. The fdhD and fdhE genes are also useful toprepare a recombinant microorganism for minimizing the production offormate as a byproduct, during the preparation of acetic acid, lacticacid or ethanol. Accordingly, the fdhD and fdhE genes according to thepresent invention will be useful in increasing the productivity ofvarious metabolites in the operation of central metabolic pathways bythe combination with a suitable metabolic pathway.

While the present invention has been described in detail with referenceto specific features, it will be apparent to those skilled in the artthat this description is illustrative only of a preferred embodiment andis not intended in any way to limit the scope of the present invention,as defined by the appended claims and equivalents thereof.

1. A gene encoding a formate dehydrogenase, selected from the groupconsisting of: a/fdhD gene encoding a formate dehydrogenase D having anamino acid sequence of SEQ ID NO: 7; and a fdhE gene encoding a formatedehydrogenase E having an amino acid sequence of SEQ ID NO:
 8. 2. ThefdhD gene according to claim 1, which has a DNA sequence of SEQ ID NO:5.
 3. The fdhE gene according to claim 1, which has a DNA sequence ofSEQ ID NO: 6
 4. A formate dehydrogenase selected from the groupconsisting of: a formate dehydrogenase D having an amino acid sequenceof SEQ ID NO: 7, which has the activity of converting formate to carbondioxide and hydrogen; and a formate dehydrogenase E having an amino acidsequence of SEQ ID NO: 8, which has the activity of converting formateto carbon dioxide and hydrogen.
 5. A recombinant vector containing atleast one gene according to claim
 1. 6. The recombinant vector accordingto claim 5, which is pMExfdhDE.
 7. A recombinant microorganism obtainedby introducing the recombinant vector according to claim 5 into a hostcell selected from the group consisting of bacteria, yeast and mold. 8.The recombinant microorganism according to claim 7, which is Mannheimiasucciniciproducens MBEL55E.
 9. The recombinant microorganism accordingto claim 7, wherein the host cell is an ethanol-producing microorganism.10. A method for preparing succinic acid, the method comprising thesteps of: culturing the recombinant microorganism according to claim 8;and recovering succinic acid from the culture broth of the recombinantmicroorganism.